ATP is released to the basolateral side of the urinary bladder epithelium during stretch and binds to purinoceptors on sub-epithelial sensory afferent neurons. According to one theory for the pathogenesis of interstitial cystitis (IC), epithelial ATP release becomes elevated and this contributes to inflammation and pain. Little information is available regarding the mechanism of mechanically-induced ATP release from transitional epithelia or its modulation. We will explore the ATP hypothesis by developing new approaches to study ATP release from acutely-isolated rat urinary bladder epithelial cells and cells maintained in primary culture. Bladder epithelial cells will also be cultured in hollow fiber bioreactors so that other epithelially-derived factors that may be co-released with ATP can be collected and identified. To firmly establish exocytosis as the primary release mechanism in uroepithelium, the involvement of SNAREs, intracellular free [Ca], vesicular pH, phosphatidyl 4,5-bisphosphate, and protein kinases will be explored. Molecular aspects of mechanically-induced ATP release and its regulation will be studied using three approaches: 1) release will be monitored macroscopically when cells are distended in a luminometer, 2) ATP release will be monitored microscopically by counting single photons to image extracellular luciferase bioluminescence, and 3) total internal reflection fluorescence imaging of single exocytotic fusions will be used. Force will be applied to single cells to induce ATP release or vesicle fusion. To identify molecular components involved in bladder epithelial force transduction and exocytosis, cultured cells will be transfected with dominant negative constructs and small interfering RNAs to inactivate key proteins in the cytoskeleton, cell adhesion complexes and vesicle trafficking machinery. Finally, maneuvers that influence ATP release in vitro will be studied in rats by recording from afferent sensory neurons that innervate the rat urinary bladder. These studies will provide new insights into the mechanism and regulation of uroepithelial ATP release, and may identify other factors that are co-released in interstitial cystitis. Understanding the molecular basis of mechano-sensitive ATP release may suggest molecular targets for therapeutic intervention.